Acridone compounds

ABSTRACT

A class of acridone compounds has been discovered that exhibits chemosensitizing and antiparasitic activity. Described herein are pharmaceutical compositions and methods for their use to treat parasitic infections, such as malaria and toxoplasmosis, and to sensitize resistant cells, such as multidrug resistant cells to other therapeutic agents. The pharmaceutical compositions and methods may also be used to treat and/or prevent psychotic diseases such as schizophrenia.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/623,433, filed Feb. 16, 2015, which is a continuation of U.S. patentapplication Ser. No. 14/066,565, filed Oct. 29, 2013, now issued as U.S.Pat. No. 8,987,296, which is a divisional of U.S. patent applicationSer. No. 12/312,503, filed May 12, 2009, now issued as U.S. Pat. No.8,592,447 on Nov. 26, 2013, which is a 35 U.S.C. §371 National Stage ofInternational Application No. PCT/US07/84560, filed Nov. 13, 2007, whichin turn claims the benefit of the earlier filing date of U.S.Provisional Application No. 60/858,802, filed Nov. 13, 2006. Allapplications are incorporated herein in their entirety.

FIELD

This disclosure concerns acridone compounds, compositions and methodsfor their use as antiparasitic agents, anti-psychotic agents, andchemosensitizers.

BACKGROUND

Throughout human history malaria has plagued mankind. Malaria remainsthe single most devastating parasitic infectious agent in the world,particularly in the developing and tropical world. Malaria infectshundreds of millions and kills roughly 2 million people each year.Globally the situation is worsening, largely due to the emergence ofmultidrug resistant strains of the responsible parasite.

In the past, the inexpensive, effective and orally availableantimalarial drug, chloroquine, was the “gold standard” treatment.Unfortunately, certain Plasmodium sp. strains have evolved resistance tochloroquine. In fact, the spread of chloroquine-resistant Plasmodium sp.parasites has rendered chloroquine almost useless for malaria treatment.Multidrug resistant strains no longer susceptible to quinoline andanti-folate-based antimalarials are common in Southeast Asia and someparts of Africa. In addition. Plasmodium sp. resistance to otherantimalarial drugs, such as artemisinin and its derivatives, has beenreported. These are particularly devastating problems in manyimpoverished parts of the world where such drugs are most needed.

Multidrug resistance is a phenomenon which has been observed in cancerand in and other conditions, such as bacterial, viral, protozoal, andfungal diseases. Multidrug resistance is a particular problem indiseases such as malaria, tuberculosis, Entamoeba histolytica (amoebicdysentery), trypanosomiasis (African sleeping sickness), leishmaniasisand AIDS pneumonia, among others. A number of diverse drugs have beenfound effective against such diseases, but in multidrug resistance adisease becomes resistant to a variety of drugs to which it initiallywas susceptible. In many examples, multidrug resistance renders drugsthat worked initially totally ineffective. Thus, there is a need notonly for new antimalarial drugs, but also for new drugs to treatmultidrug resistance.

Parasitic diseases have also been associated with psychotic diseasessuch as schizophrenia. In other words, parasitic infection in a subjectcould result in schizophrenia and schizophrenic symptoms. There exists acontinued need for anti-psychotic drugs.

SUMMARY

This disclosure concerns the discovery of a class of acridone compounds.This class of compounds exhibits chemosensitizing and antiparasiticactivity. Exemplary chemosensitizing and antiparasitic acridonesdisclosed herein include those represented by the formula:

or a pharmaceutically acceptable salt thereof;

wherein R is H, —R²NR³R⁴ or —R¹G;

X is H, halogen, haloalkyl, OR⁵ or —YR¹G:

n is 0-4;

Y is H, —CH₂—, —CH₂O—, —O—, —N(R⁶)— or —S—;

R⁵ is lower alkyl, haloalkyl or —R⁷NR⁸R⁹;

R¹, R² and R⁷ independently are optionally substituted alkyl;

G is —NR¹⁰R¹¹, halogen or fluoroalkyl;

R³, R⁴, R⁶, R⁸ and R⁹ independently are H, lower alkyl, or aralkyl;

R¹⁰ and R¹¹ independently are H, lower alkyl, aralkyl or together forman aliphatic or aromatic ring optionally including one or moreadditional heteroatoms; and when n is 0, R is —R¹G.

Also described are hydrates and pharmaceutically acceptable prodrugs andsalts of the acridones above. Moreover, all enantiomeric, diastereomericand geometric isomeric forms of the disclosed formulas are intended.

Also described are uses of the compounds and methods of administration.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 demonstrates the inhibition of in vitro heme aggregation byxanthone 2,3,4,5,6-pentahydroxyxanthone; panel B illustrates aspectrophotometric titration curve of the heme:3,6-bis-ε-(N,N-diethylamino)-amyloxy xanthone (C5) complex.

FIG. 2 illustrates the in vivo antimalarial activity of a disclosedantimalarial acridone (T2) against P. yoelii (K) in female CF-1 mice ina 4 day suppressive trial.

FIG. 3 illustrates the in vivo curative efficacy of a single ig dose ofexemplary acridones vs. P. yoelii (K) patent infection, determined 48and 72 hours after treatment.

FIG. 4 includes isobolograms charting the efficacy of selectedquinolines in combination with the antimalarial acridone T3.5 in vitroagainst multi drug resistant (MDR) P. falciparum (Dd2), wherein valuesbelow the diagonal (additive) line indicate synergism.

FIG. 5 is an isobologram charting the efficacy of quinine in combinationwith the antimalarial acridones T2 (top line) or T3.5 (bottom line) invitro against MDR P. falciparum Dd2.

FIG. 6 is an isobologram charting the efficacy of quinine in combinationwith T3.5 in vitro against CQ-sensitive (D6 top line) and MDR (Dd2bottom line) P. falciparum demonstrating synergy of the combination,particularly against MDR P. falciparum.

FIG. 7 is a graph showing in vivo antimalarial activity of acridones T2and T3.5 as well as quinine against P. yoelii.

FIG. 8 (panels A-E) shows metabolite profiles of the acridone T3.5 inmurine S-9 with NADPH for 60 minutes.

DETAILED DESCRIPTION

Disclosed herein are recently discovered acridone compounds that arehighly effective in reversing multidrug resistance and for directlykilling and/or inhibiting the growth of parasites, including multi-drugresistant parasites. Such compounds may be used, at least, inpharmaceutical compositions, and to treat parasitic diseases such asmalaria and toxoplasmosis. The compounds may be used to inhibit thegrowth of organisms such as Plasmodium sp., Toxoplasma gondii,Mycobacterium tuberculosis, Pneumocystis carinii. In yet otherembodiments, any of the foregoing or other disclosed compounds can beincorporated into pharmaceutical compositions that include atherapeutically effective amount of the compound or extract, and apharmaceutically acceptable carrier. In some instances, a disclosedpharmaceutical composition further includes at least one additionalactive agent. In certain embodiments the second active agent is anantimalarial therapeutic agent (such as a quinoline compound, e.g.quinine, chloroquine, mefloquine or the like, a peroxide compound, suchas an artemisinin, a folate synthesis inhibitor, such as sulfadoxineand/or pyrimethamine, or a cinchona alkaloid, such as quinine, quinidineand the like).

The present disclosure also concerns methods of treating a subject forparasitemia, such as malaria or Plasmodium sp. infection byadministering to the subject a therapeutically effective amount (such asfrom about 1 to about 50 mg/kg) of any of the compounds disclosedherein. In some cases, the compound is administered prophylactically. Inother embodiments, the malarial pathogen is P. falciparum.

Also disclosed are methods for inhibiting the growth of Plasmodium sp.involving contacting at least one Plasmodium sp. parasite with a growthinhibitory amount (such as from about 0.1 to about 500 nM, such as fromabout 1 to about 250 nM, in particular from about 5 to about 50 nM) ofat least one disclosed compound. In some method embodiments, thePlasmodium sp. is P. falciparum, P. vivax, P. ovale, or P. malariae, ora combination thereof.

The disclosed compounds may be used to inhibit the growth of protozoasuch as Toxoplasma gondii, bacteria such as Mycobacterium tuberculosis,and fungal parasites such as Pneumocystis carinii. Treating diseasescaused by these parasites is also within the scope of this disclosure.Also encompassed are diseases and conditions caused by Toxoplasma sp.,Mycobacterium sp., and Pneumocystis sp.

In some cases the disclosed acridone compounds are used aschemosensitizers to sensitize a resistant cell, for example by reversalof multidrug resistance. Accordingly, further disclosed embodimentsinclude methods for potentiating a drug that has been rendered lesseffective by resistance, such as multidrug resistance. In these methodsa disclosed acridone compound is administered in conjunction with thedrug to be potentiated. Classes of drugs whose efficacy can be restoredusing the disclosed acridones as chemosensitizers include, withoutlimitation, anticancer agents, antibiotics, antiparasitics, antifungalsand antivirals.

I. Terms:

The following explanations of terms and methods are provided to betterdescribe the present compounds, compositions and methods, and to guidethose of ordinary skill in the art in the practice of the presentdisclosure. It is also to be understood that the terminology used in thedisclosure is for the purpose of describing particular embodiments andexamples only and is not intended to be limiting.

As used herein, the singular terms “a,” “an,” and “the” include pluralreferents unless context clearly indicates otherwise. Similarly, theword “or” is intended to include “and” unless the context clearlyindicates otherwise. Also, as used herein, the term “comprises” means“includes.” Hence “comprising A or B” means including A, B, or A and B.

“Optional” or “optionally” means that the subsequently described eventor circumstance can but need not occur, and that the descriptionincludes instances where said event or circumstance occurs and instanceswhere it does not.

“Derivative” refers to a compound or portion of a compound that isderived from or is theoretically derivable from a parent compound.

“Subject” includes, without limitation, humans and veterinary subjects,particularly economically important animals, such as livestock andavians, particularly poultry infected with protozoans, such as Eimeria.

The phrase “treating a disease” refers to inhibiting the fulldevelopment of a disease or condition, for example, in a subject who isat risk for a disease such as trypanasomal infection, for instance a P.falciparum infection, particularly a multidrug resistant strain of P.falciparum. Other instances of diseases include those caused by T.gondii, M. tuberculosis, and P. carinii. The phrase “treating a disease”also encompasses diminishing or reversing multidrug resistance, tosensitize a pathogen to a drug to which it has acquired resistance.Multiple drug resistance occurs when target cells, including trypanosomecells, such as P. falciparum become resistant to a drug being usedduring treatment and to other drugs that are different and structurallyunrelated to the drug being administered. Certain compounds, including,without limitation verapamil, diltiazem, cyclosporin and catharanthineare known to attenuate or reverse drug resistance in some cells. Suchcompounds are referred to as “chemosensitizers” or “reversal agents.”

“Treatment” refers to a therapeutic intervention that ameliorates a signor symptom of a disease or pathological condition after it has begun todevelop. As used herein, the term “ameliorating,” with reference to adisease or pathological condition, refers to any observable beneficialeffect of the treatment. The beneficial effect can be evidenced, forexample, by a delayed onset of clinical symptoms of the disease in asusceptible subject, a reduction in severity of some or all clinicalsymptoms of the disease, a slower progression of the disease, animprovement in the overall health or well-being of the subject, or byother parameters well known in the art that are specific to theparticular disease.

A “prophylactic” treatment is a treatment administered to a subject whodoes not exhibit signs of a disease or exhibits only early signs for thepurpose of decreasing the risk of developing pathology.

By the term “coadminister” is meant that each of at least two compoundsbe administered during a time frame wherein the respective periods ofbiological activity overlap. Thus, the term includes sequential as wellas coextensive administration of two or more drug compounds.

In one embodiment a “chemosensitizer” or “chemosensitizing agent” or“reversing agent” refers to an agent that diminishes or abolishesresistance to a therapeutic agent. In one embodiment, achemonusensitizer allows the net accumulation of a therapeutic compoundin multidrug resistant cells. In some examples, chemosensitizersdisclosed herein are effective to result in the accumulation of atherapeutic compound to an equivalent level to the net accumulation ofthe therapeutic compound in non-multidrug resistant cells. The presenceof a chemosensitizer may also merely increase the amount of thetherapeutic compound able to accumulate in a multidrug resistant cellcompared to the amount accumulated in the absence of thechemosensitizer. The chemosensitizers disclosed herein also operate toreverse other mechanisms of resistance besides multidrug resistance.

The term “neoplasm” refers to an abnormal cellular proliferation, whichincludes benign and malignant tumors, as well as other proliferativedisorders.

The term “acyl” refers group of the formula RC(O)— wherein R is anorganic group.

The term “alkyl” refers to a branched or unbranched saturatedhydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl,octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like. A“lower alkyl” group is a saturated branched or unbranched hydrocarbonhaving from 1 to 10 carbon atoms.

The term “alkenyl” refers to a hydrocarbon group of 2 to 24 carbon atomsand structural formula containing at least one carbon-carbon doublebond.

The term “alkynyl” refers to a hydrocarbon group of 2 to 24 carbon atomsand a structural formula containing at least one carbon-carbon triplebond.

The terms “halogenated alkyl” or “haloalkyl group” refer to an alkylgroup as defined above with one or more hydrogen atoms present on thesegroups substituted with a halogen (F, Cl, Br, I).

The term “cycloalkyl” refers to a non-aromatic carbon-based ringcomposed of at least three carbon atoms. Examples of cycloalkyl groupsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, and the like. The term “heterocycloalkyl group” is acycloalkyl group as defined above where at least one of the carbon atomsof the ring is substituted with a heteroatom such as, but not limitedto, nitrogen, oxygen, sulfur, or phosphorous.

The term “aliphatic” is defined as including alkyl, alkenyl, alkynyl,halogenated alkyl and cycloalkyl groups as described above. A “loweraliphatic” group is a branched or unbranched aliphatic group having from1 to 10 carbon atoms.

The term “aryl” refers to any carbon-based aromatic group including, butnot limited to, benzene, naphthalene, etc. The term “aromatic” alsoincludes “heteroaryl group,” which is defined as an aromatic group thathas at least one heteroatom incorporated within the ring of the aromaticgroup. Examples of heteroatoms include, but are not limited to,nitrogen, oxygen, sulfur, and phosphorous. The aryl group can besubstituted with one or more groups including, but not limited to,alkyl, alkynyl, alkenyl, aryl, halide, nitro, amino, ester, ketone,aldehyde, hydroxy, carboxylic acid, or alkoxy, or the aryl group can beunsubstituted. The term “alkyl amino” refers to alkyl groups as definedabove where at least one hydrogen atom is replaced with an amino group.

“Carbonyl” refers to a radical of the formula —C(O)—.Carbonyl-containing groups include any substituent containing acarbon-oxygen double bond (C═O), including acyl groups, amides, carboxygroups, esters, ureas, carbamates, carbonates and ketones and aldehydes,such as substituents based on —COR or —RCHO where R is an aliphatic,heteroaliphatic, alkyl, heteroalkyl, hydroxyl, or a secondary, tertiary,or quaternary amine.

“Carboxyl” refers to a —COOH radical. Substituted carboxyl refers to—COOR where R is aliphatic, heteroaliphatic, alkyl, heteroalkyl, or acarboxylic acid or ester.

The term “hydroxyl” is represented by the formula —OH. The term “alkoxygroup” is represented by the formula —OR, where R can be an alkyl group,optionally substituted with an alkenyl, alkynyl, aryl, aralkyl,cycloalkyl, halogenated alkyl, or heterocycloalkyl group as describedabove.

The term “hydroxyalkyl” refers to an alkyl group that has at least onehydrogen atom substituted with a hydroxyl group. The term “alkoxyalkylgroup” is defined as an alkyl group that has at least one hydrogen atomsubstituted with an alkoxy group described above.

The term “lower alcohol” refers to an alkyl group containing from one toten carbon atoms substituted with one or more hydroxy (—OH) moieties.Examples of lower alcohols include, without limitation

The term “amine” or “amino” refers to a group of the formula —NRR′,where R and R′ can be, independently, hydrogen or an alkyl, alkenyl,alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, orheterocycloalkyl group described above.

The term “amide group” is represented by the formula —C(O)NRR′, where Rand R′ independently can be a hydrogen, alkyl, alkenyl, alkynyl, aryl,aralkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl groupdescribed above.

The term “aralkyl” refers to an aryl group having an alkyl group, asdefined above, attached to the aryl group. An example of an aralkylgroup is a benzyl group.

Optionally substituted groups, such as “substituted alkyl,” refers togroups, such as an alkyl group, having from 1-5 substituents, typicallyfrom 1-3 substituents, selected from alkoxy, optionally substitutedalkoxy, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, aryl,carboxyalkyl, optionally substituted cycloalkyl, optionally substitutedcycloalkenyl, optionally substituted heteroaryl, optionally substitutedheterocyclyl, hydroxy, thiol and thioalkoxy.

The term “protecting group” or “blocking group” refers to any group thatwhen bound to a functional group.

Reference will now be made in detail to the presently preferredcompounds.

II. Chemosensitizing and Antiparasitic Compounds:

In general the acridone compounds disclosed herein can be represented bythe formula

or a pharmaceutically acceptable salt thereof;

wherein R is H, —R²NR³R⁴ or —R¹G;

X is H, halogen, haloalkyl, OR⁵ or —YR¹G;

n is 0-4;

Y is H, —CH₂—, —CH₂O—, —O—, —N(R⁶)— or —S—;

R⁵ is lower alkyl, haloalkyl or —R⁷NR⁸R⁹;

R¹, R² and R⁷ independently are optionally substituted alkyl;

G is —NR¹⁰R¹¹, halogen or fluoroalkyl;

R³, R⁴, R⁶, R⁸ and R⁹ independently are H, lower alkyl, or aralkyl;

R¹⁰ and R¹¹ independently are H, lower alkyl, aralkyl or together forman aliphatic or aromatic ring optionally including one or moreadditional heteroatoms; and when n is 0, R is —R¹G.

Compounds of the formula above can include, on an aryl ring, from 1 to 4X groups and from 0 to 4 —YR¹G groups. In a preferred embodiment of thedisclosed acridone compounds n is from 1 to 4. In a still more preferredembodiment of the disclosed acridone compounds n is 1, and the compoundshave a —YR¹G group at only one of positions 1 to 4 on the acridone ringsystem. In another embodiment, the disclosed acridone compounds have theformula

wherein m is from 2 to 10. In a further embodiment, both aryl rings beara YR¹G moiety, examples of such compounds can be represented by theformula

In further embodiments R can represent —R¹G, with examples of suchcompounds having the formula

Particular embodiments of these compounds also are represented by theformula

Disclosed acridones include, in some embodiments, those having R¹ be abranched alkyl group or include a cycloalkyl group. For example, R¹optionally is substituted with a lower alkyl group, such as a methyl,ethyl, propyl or butyl group. In other examples R^(L) is substitutedwith a hydroxy, alkoxy, lower alkyl or halo group. R¹ also can include acyclic group, for example a cycloalkyl group.

In other embodiments, R¹ is an alkyl chain. For example, —YR¹G canrepresent —Y(CH₂)_(n)G with n being from 2 to 10 or from 2 to 5.Particular examples of such compounds include those having the formula

with additional examples having the formula

with n and m independently being from 2 to 10, such as from 2 to 5.

In particular disclosed acridone compounds having the formulas presentedabove, G represents —NR¹⁰R¹¹. Typically, R¹⁰ and R¹¹ independently areselected from H, lower alkyl, such as methyl, ethyl, propyl, butyl andthe like; aralkyl, such as benzyl; or together form an aliphatic oraromatic ring optionally including one or more additional heteroatoms.Such cyclic groups can include, in addition to the nitrogen atom, one ormore additional heteroatoms, such as an additional nitrogen, oxygenand/or sulfur atom. Specific cyclic groups represented by G include,without limitation pyrrolidino, pyridine, piperidino, morpholino,piperazino, imidazolyl, pyrazolyl or triazolyl moieties.

In certain embodiments R¹⁰ and R¹¹ are the same, such as in exemplarycompounds wherein —R¹G represents —CH₂CH₂N(Et)₂. Particular examples ofsuch N,N-diethyl substituted acridone compounds have the formula:

In other embodiments, R¹⁰ is H and R¹¹ represents —C(CH)₃. Particularexamples have the formula:

In some instances G is haloalkyl, such as bromo, chloro or fluoroalkyl.Typically, when G is a fluoroalkyl moiety. G includes at least onetrifluoromethyl group. Particular examples of such haloalkyl compoundshave the formula:

Certain disclosed acridone compounds include a halo and/or a fluoroalkylmoiety at one or more of positions 5 to 8 on the acridone ring.Particular examples of such compounds include, without limitation, the6-chloro compounds having the formula:

and trifluoromethyl substituted compounds of the formula:

Further such compounds include a halo and/or a fluoroalkyl moiety at twoor more of positions 5 to 8 on the acridone ring. Exemplary compoundscan be represented by the formula:

with particular embodiments including compounds of the formula:

wherein X and X′ independently are selected from H, halogen, haloalkyl,OR⁵ or —YR¹G. In certain examples both X and X′ are halogens, such assubstituents.

Still other embodiments of the disclosed antiparasitic andchemosensitizing acridones are represented by the formulas:

Particular examples of acridones disclosed herein include, withoutlimitation, those of the formulas:

Exemplary compounds also are referred to herein by their chemical names.Such names include, without limitation,3-(2-diethylaminoethoxy)-9-acridone;3-(2-diethylaminoethoxy)-6-chloro-9-acridone;2-(2-diethylaminoethoxy)-6-chloro-9-acridone;3-(3-diethylaminopropoxy)-9-acridone;3-(5-diethylaminopentyloxy)-6-chloro-9-acridone;2-(2-diethylaminoethoxy)-6-chloro-10-(2-diethylaminoethyl)-9-acridone;3-(2-diethylaminoethoxy)-6-chloro-10-(2-diethyl-aminoethyl)-9-acridone;and3-(3-tert-Butylamino-propyloxy)-10-(3-tert-butylamino-propyl)-5,7-dichloro-9-acridone.

Also contemplated are pharmaceutically acceptable salts and prodrugs ofthe compounds described above. The term “pharmaceutically acceptablesalt or prodrug” is used throughout the specification to describe anypharmaceutically acceptable form (e.g., ester, phosphate ester, salt ofan amino or related group) of an acridone compound, which, uponadministration to a subject, provides or produces an active compound.Pharmaceutically acceptable salts include those derived frompharmaceutically acceptable inorganic or organic bases and acids. Inparticular, suitable salts include those derived from alkali metals suchas potassium and sodium, alkaline earth metals such as calcium andmagnesium, among numerous other acids well known in the pharmaceuticalart.

Particular disclosed acridone compounds possess at least one basic group(and typically plural basic groups) that can form acid-base salts withacids. Examples of basic groups include, but are not limited to, anamino group or imino group. Examples of inorganic acids that can formsalts with such basic groups include, but are not limited to, mineralacids such as hydrochloric acid, hydrobromic acid, sulfuric acid orphosphoric acid. Basic groups also can form salts with organiccarboxylic acids, sulfonic acids, sulfo acids or phospho acids orN-substituted sulfamic acid, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, gluconicacid, glucaric acid, glucuronic acid, citric acid, benzoic acid,cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid,2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinicacid or isonicotinic acid, and, in addition, with amino acids, forexample with α-amino acids, and also with methanesulfonic acid,ethanesulfonic acid, 2-hydroxymethanesulfonic acid,ethane-1,2-disulfonic acid, benzenedisulfonic acid,4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate or N-cyclohexylsulfamic acid(with formation of the cyclamates) or with other acidic organiccompounds, such as ascorbic acid.

Also disclosed are pharmaceutically acceptable prodrugs of acridonecompounds. Pharmaceutically acceptable prodrugs refer to compounds thatare metabolized, for example, hydrolyzed or oxidized, in the subject toform an antiparasitic or chemosensitizing compound of the presentdisclosure. Typical examples of prodrugs include compounds that have oneor more biologically labile protecting groups on or otherwise blocking afunctional moiety of the active compound. Prodrugs include compoundsthat can be oxidized, reduced, aminated, deaminated, hydroxylated,dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated,acylated, deacylated (such as deesterified), phosphorylated,dephosphorylated to produce the active compound. Exemplary acridonecompounds possess activity against a parasite and/or resistant cell, orare metabolized to a compound that exhibits such activity.

The term “prodrug” also is intended to include any covalently bondedcarriers that release an active parent drug of the present invention invivo when the prodrug is administered to a subject. Since prodrugs oftenhave enhanced properties relative to the active agent pharmaceutical,such as solubility and bioavailability, the compounds disclosed hereincan be delivered in prodrug form. Thus, also contemplated are prodrugsof the presently claimed compounds, methods of delivering prodrugs andcompositions containing such prodrugs. Prodrugs of the disclosedcompounds typically are prepared by modifying one or more functionalgroups present in the compound in such a way that the modifications arecleaved, either in routine manipulation or in vivo, to yield the parentcompound. Prodrugs include compounds having a hydroxy, amino, orsulfhydryl group functionalized with any group that is cleaved to yieldthe corresponding hydroxyl, free amino, or free sulfhydryl group,respectively. Examples of prodrugs include, without limitation,compounds having a hydroxy, amino and/or sulfhydryl group acylated withan acetate, formate, and/or benzoate group. Various forms of prodrugsare known in the art, for example, as discussed in Bundgaard, (ed.),Design of Prodrugs, Elsevier (1985); Widder, et al. (ed.), Methods inEnzymology, vol. 4, Academic Press (1985); Krogsgaard-Larsen. et al.,(ed.). Design and Application of Prodrugs, Textbook of Drug Design andDevelopment, Chapter 5, 113 191 (1991), Bundgaard, et al., Journal ofDrug Delivery Reviews, 8:1 38(1992), Bundgaard. J. of PharmaceuticalSciences, 77:285 et seq. (1988); and Higuchi and Stella (eds.) Prodrugsas Novel Drug Delivery Systems, American Chemical Society (1975).

Protected derivatives of the disclosed acridone compounds also arecontemplated. A variety of suitable protecting groups for use with thedisclosed compounds are disclosed in Greene and Wuts Protective Groupsin Organic Synthesis; 3rd Ed.: John Wiley & Sons, New York (1999).Particular examples of protected derivatives include acridone compoundsin which the 9-keto group is converted to an alkoxy group.

It is understood that substituents and substitution patterns of thecompounds described herein can be selected by one of ordinary skill inthe art to provide compounds that are chemically stable and that can bereadily synthesized by techniques known in the art and further by themethods set forth in this disclosure.

The compounds described herein may possess one or more asymmetriccenters; such compounds can therefore be produced as individual (R)- or(S)-stereoisomers or as mixtures thereof. Unless indicated otherwise,the description or naming of a particular compound in the specificationand claims is intended to include both individual enantiomers andmixtures, racemic or otherwise, thereof. The methods for thedetermination of stereochemistry and the separation of stereoisomers arewell-known in the art (see, e.g., March, Advanced Organic Chemistry, 4thedition, John Wiley and Sons, New York, 1992, Chapter 4).

Additionally, the structural formulas herein are intended to cover,where applicable, solvated as well as unsolvated forms of the compounds.Such solvates refer to a pharmaceutically acceptable form of a specifiedcompound complexed with a solvent molecule, the solvate retaining thebiological effectiveness of the compound. Examples of solvates include,by way of example, hydrates and compounds complexed with other solvents,such as isopropanol, ethanol, methanol, dimethyl sulfoxide, ethylacetate and/or acetone.

III. Compositions and Methods:

Another aspect of the disclosure includes pharmaceutical compositionsprepared for administration to a subject and which include atherapeutically effective amount of one or more of the currentlydisclosed compounds. Disclosed also are methods for administering thedisclosed compounds and compositions. The therapeutically effectiveamount of a disclosed compound will depend on the route ofadministration, the type of mammal that is the subject and the physicalcharacteristics of the subject being treated. Specific factors that canbe taken into account include disease severity and stage, weight, dietand concurrent medications. The relationship of these factors todetermining a therapeutically effective amount of the disclosedcompounds is understood by those of ordinary skill in the art.

The compounds disclosed herein may be administered orally, topically,transdermally, parenterally, via inhalation or spray and may beadministered in dosage unit formulations containing conventionalnon-toxic pharmaceutically acceptable carriers, adjuvants and vehicles.

Typically, oral administration or administration intravenously, such asvia injection is preferred. However the particular mode ofadministration employed may be dependent upon the particular disease,condition of patient, toxicity of compound and other factors as will berecognized by a person of ordinary skill in the art.

Pharmaceutical compositions for administration to a subject can includecarriers, thickeners, diluents, buffers, preservatives, surface activeagents and the like in addition to the molecule of choice.Pharmaceutical compositions can also include one or more additionalactive ingredients such as antimicrobial agents, anti-inflammatoryagents, anesthetics, and the like. Pharmaceutical formulations caninclude additional components, such as carriers. The pharmaceuticallyacceptable carriers useful for these formulations are conventional.Remington's Pharmaceutical Sciences, by E. W. Martin, Mack PublishingCo., Easton, Pa., 19th Edition (1995), describes compositions andformulations suitable for pharmaceutical delivery of the compoundsherein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually contain injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (for example, powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

In certain embodiments the compounds used in the method are provided arepolymorphous. As such, the compounds can be provided in one or morephysical form, such as different crystal forms, crystalline, liquidcrystalline or non-crystalline (amorphous) forms.

Also disclosed herein is a method of inhibiting the growth of amicrobial pathogen, such as a parasite, particularly a protozoanparasite, such as those responsible for diseases such as malaria,trypanosomiasis, Chagas' disease, leishmaniasis, giardiasis, andamoebiasis. Also discloses are methods for the inhibition of diseasescaused by Toxoplasma sp., Mycobacterium sp., and Pneumocystis sp. Themethod comprises providing an effective amount of an acridone compoundto inhibit pathogen growth in vivo or in vitro.

In one aspect, the method can be used to treat a subject having amicrobial infection. The method comprises administering to the subject atherapeutically effective amount of a disclosed acridone compound, suchas at least about 0.005 mg/kg. In certain embodiments, the acridone isadministered at a dose of at least about 0.02 mg/kg. Typically, no morethan about 250 mg/kg of the agent is administered, and more typicallyless than about 50 mg/kg, and even more typically less than about 10mg/kg such as about 5 mg/kg or about 0.2 mg/kg. Hence in certainembodiments the dosage range is about 0.005 to about 10 mg/kg, or about0.02 to about 5 mg/kg, or about 0.2 to about 5 mg/kg, such as from about1 to about 5 mg/kg.

Certain disclosed compounds inhibit the aggregation of heme. A number ofpathogens, including trypanosomes, such as Plasmodium, a causative agentof malaria, degrade hemoglobin to obtain amino acids, and in so doingliberate toxic heme. To avoid the toxic effects of the liberated heme,these pathogens have evolved a mechanism for “aggregation” of heme unitsto form hemozoin. Thus, without limitation to theory, the disclosedacridone inhibitors of heme aggregation may exert their antiparasiticeffect by blocking heme aggregation and preventing these organisms fromgaining access to the host's supply of heme iron, or by causing abuild-up of toxic levels of heme in the organism.

The disclosed acridone compounds are particularly effective when usedwith one or more other agents or therapies useful in the treatment ofresistant disorders, such as disorders caused by multidrug resistantcells. For example, one or more disclosed acridones can be administeredin combination with effective doses of other medicinal andpharmaceutical agents, or in combination other non-medicinal therapies,such as hormone or radiation therapy. The term “administration incombination with” refers to both concurrent and sequentialadministration of the active agents.

In one embodiment the disclosed acridone compounds are used in a methodof enhancing the intracellular accumulation of a drug in multidrugresistant cells wherein the accumulation depends upon inhibitingtransport by the multidrug resistance transport system involvingP-glycoprotein. In such methods, the compounds of the present inventionare coadministered with the drug.

The administration may be in vitro or in vivo. In some embodiments, theenhancement of accumulation of the drug in multidrug resistant cells isin vivo. In particular embodiments, one or more disclosed acridonecompound is administered to a cancer patient to treat a tumor that hasbecome multidrug resistant in the course of therapy. Typically, in suchembodiments, chemotherapeutic agents are administered with the compoundsof the present invention. The coadministration is designed to enhanceaccumulation of the agent following reversal of the multidrug resistantphenotype by interaction of the compounds of the present invention withthe multidrug resistance transport system. Thus, the coadministration isdesigned to cause the chemotherapeutic agent to accumulate in amountseffective for cytotoxicity, whereas when the agent is administeredalone, accumulation in effective amounts does not occur. Thiscoadministration regimen can be applied to any cell which exhibits themultidrug resistance phenotype, for example, as a result ofoverexpression of the multidrug resistance protein, e.g.,P-glycoprotein.

In certain cases, the disclosed acridones in combination with at leastone other therapeutic agent exhibit a synergistic effect. Synergy isobserved when the agents administered have a greater than additiveeffect when administered in combination. In other embodiments thedisclosed acridones exhibit synergy with one or more antitumor,antiviral, antiparasitic (such as anti-trypanosomal, such asantimalarial) or antibiotic agent. Thus, the disclosed acridones areused in combination to treat viral infections, such as retroviral, forexample HIV infections; neoplasms, particularly malignant neoplasms;bacterial infections, such as S. aureus, S. epidernnidis, S. pneumoniae,E. faecalis, E. faecium, and drug resistant Gram positive cocci, such asmethicillin-resistant staphylococci and vancomycin-resistantenterococci; and parasitic infections, such as trypanosomal infections,for example malaria, schistosomiasis, toxoplasmosis, and leishmaniasis.In particular embodiments, the acridone compounds exhibit, incombination with a second therapeutic agent a fixed-ratio concentrationsynergism of less than about 1, such less than about 0.8, particularlyless than about 0.5.

in particular the disclosed acridones are effective against malariaand/or Plasmodium sp. parasitemia either alone or in combination withone or more additional antimalarial agents or therapies. In someexamples, the one or more antimalarial agents or therapies for use incombination with the disclosed acridones include artesunate andmefloquine (either individually or in an artesunate-mefloquinecombination), or sulfadoxine and pyrimethamine (either individually orin a sulfadoxine-pyrimethamine combination (commercially available asFANDISAR)). In particular examples, the one or more other antimalarialagents or therapies have at least one different mode of action than isproposed for a disclosed acridone; thus, for instance, a combinationagent or therapy may target mitochondria and/or dihydrofolate reductase.

In one embodiment the disclosed acridones can be used either alone or incombination with another drug to effect a curative treatment regiment.As used herein a curative dose refers the dose at which parasitemia iscleared for 28 days. Typically, the curative dose is administered overseveral days, such as for 1 to 10 days, such as over 1 to 7 days, suchas from 2 to 5 days, for example over 3 days. The curative dose can beadministered once to several times daily, and typically is given in aonce, twice or three times daily dosage regimen.

For malaria prevention, a typical dosing schedule could be, for example,about 2.0 to about 1000 mg/kg weekly beginning about 1 to about 2 weeksprior to malaria exposure taken up until about 1 to about 2 weekspost-exposure. The prophylactic dose also can be given in a once dailyto several times daily dosage regimen.

In certain embodiments the disclosed acridones can be used aschemosensitizing agents to restore the clinical efficacy of anantimalarial drug to which a parasite strain has required resistance.For example, in certain examples the efficacy of a drug can be restoredby administration of a disclosed acridone compound. In particular, theefficacy of quinoline based antimalarials, such as quinine, chloroquineand quinidine can be restored against resistant parasites bycoadministration with a disclosed acridone.

Although the acridones disclosed herein act as reversal agents, in oneembodiment, the disclosed acridones are used to treat a parasiticinfection, such as malaria, in combination with a sensitizing orreversal agent. In particular, reversal agents are suitable forcoadministration with the disclosed acridone compounds. By way ofexample, reversal agents suitable for coadministration with thedisclosed acridones include, without limitation, amitriptyline,amlodipine, azatadine, chlorpheniramine, citalopram, cyclosporine,cyproheptadine, cyproheptadine, desipramine,diethyl-(3-[3-(4-methoxy-benzylidene)-pyrrolidin-1-yl]-propyl)-amine,erythromycin, fantofarone, fluoxetine, haloperidol, icajine, imipramine,isoretuline, ivermectin, ketotefin, ketotifen, nomifensine, NP30:C₉H₁₉-Phenyl-(O—CH₂CH₂)₃₀OH, oxaprotiline, probenecid, progesterone,promethazine, strychnobrasiline, BG958, trifluoperazine, verapamil, orWR 268954. Other useful combinations include combination with CQ,desethyl-CQ, quinine, mefloquine or amodiaquine against multidrugresistant P. falciparum Dd2.

As noted above, in embodiments of combination therapy disclosed herein,a disclosed acridone is used to sensitize a multidrug resistant neoplasmto a chemotherapeutic agent. In one embodiment, the chemotherapeuticagent is an agent of use in treating neoplasms such as solid tumors.Examples of such chemotherapeutic agents can that can be used incombination with a disclosed acridone include microtubule bindingagents, DNA intercalators or cross-linkers, DNA synthesis inhibitors,DNA and/or RNA transcription inhibitors, antibodies, enzymes, enzymeinhibitors, gene regulators, and/or angiogenesis inhibitors. One ofskill in the art can readily identify additional chemotherapeutic agentsof use (e.g., see Slapak and Kufe, Principles of Cancer Therapy, Chapter86 in Harrison's Principles of Internal Medicine, 14th edition; Perry etal., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2^(nd) ed., ©2000 Churchill Livingstone, Inc; Baltzer L, Berkery R (eds.): OncologyPocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995;Fischer D S, Knobf M F, Durivage H J (eds.): The Cancer ChemotherapyHandbook, 4th ed. St. Louis, Mosby-Year Book, 1993).

In addition to treating diseases such as malaria, the compoundsdescribed can be used to treat other parasitic diseases such astoxoplasmosis. Toxoplasmosis is caused by a sporozoan parasite of theApicomplexa called the Toxoplasma gondii. It is a common tissue parasiteof humans and animals. Approximately 1 billion people worldwide aresero-positive for T. gondii, including roughly half of the U.S.population. Humans are merely incidental hosts for the organism, i.e.,hosts to the asexual cycle only. Most of the infections appear to beasymptomatic (90%), however toxoplasmosis poses a serious health riskfor immuno-compromised individuals, such as organ transplant recipients,cancer and AIDS patients, and the unborn children of infected mothers.Congenital toxoplasmosis occurs in about 1 in 1.000 live births witheffects ranging from asymptomatic to stillbirth, but more commonlyretinochoroiditis, cerebral calcification, psychomotor deficit, ormental retardation, and severe brain damage.

T. gondii is an obligate intracellular protozoon. There are threeinfectious stages in the developmental cycle of T. gondii, thetrophozoite (tachyzoite), the bradyzoites (tissue cyst forms), and thesporozoites that are found in oocysts. The tachyzoite presents as anactive developmental stage in which the parasites undergo multiplicationwithin a host cell. In this stage the parasite has the shape of a smallcrescent, roughly 2 by 6 μm, with a pointed anterior end and a roundedposterior end. Accumulation of numerous T. gondii within a single hostcell has been termed as the pseudocyst form. The cyst form of T. gondiiis essentially a resting, non-proliferating stage of the parasite, and atough membrane protects the organisms. Cysts range in size from 30 to100 microns, and predominate in chronic infections and accumulate inbrain, heart muscle, and the diaphragm of the host. Each cyst containsmany hundreds or thousands of organisms and although sizeable in natureand somewhat damaging to host tissues, it is interesting to note thatthere is rarely local immune reaction to the presence of the invadingorganisms or their dwellings. These cysts can reside in the human hostfor life.

The cat is the definitive host (i.e., host to the parasite sexual cycle)for T. gondii and is needed to complete its life cycle. Ingestion oftissue cysts in an intermediate host, such as a mouse or a rat, leads toinfection of the cat. The organisms penetrate epithelial cells of thesmall intestine of the cat and initiate the development of numerousgenerations of T. gondii. After a process of asexual development, thesexual cycle starts about 2 days after tissue cysts have been ingestedby the cat, yielding oocysts which are passed in the feces and picked upby animals from contaminated water or by ingestion of infected meat.

Recent scientific experiments have shown a link between infectiousagents and psychotic diseases. For example, acute infection of humanbeings with T. gondii can produce symptoms similar to schizophrenia.See, Torrey and Yolken, Toxoplasma gondii and Schizophrenia, Emerg.Infect. Dis. 9(11), 1375-1380 (2003) and Schizophr Bull. 33(3), 727-728(2007), both references incorporated herein, in their entirety, byreference. For example, mothers having antibodies to T. gondii late inpregnancy had an increased risk of giving birth to offspring who laterdeveloped a schizophrenia spectrum disorder. Other studies have foundthat newborns that are sero-positive for antibodies to T. gondii have anincreased risk of later being diagnosed with schizophrenia.Schizophrenia is a debilitating disease of the mind and has beendescribed as one of the worst diseases to affect mankind and one of themost expensive diseases to treat. It affects about 1% of the populationof the United States.

In humans, toxoplasmosis is generally treated by a combination ofsulfonamides and pyrimethamine. Although these drugs are helpful inmanagement of the acute stage of disease, they usually do not eradicateinfection and treatment failure rates of 20 to 50% have been reported.Atovaquone, a second line therapeutic for treatment of toxoplasmosis, isthe only drug known to have some activity against the dormant cyststage; it is believed to target the parasite respiratory pathway at thecytochrome bc₁ complex. Efficacy of atovaquone is limited by itsvariable intestinal absorption.

The compounds described herein may be used as an adjunct to, orreplacement of, drugs such as atovaquone. Treatment of parasiticinfections such as toxoplasmosis by using the compounds and methodsdescribed herein, is within the scope of this disclosure. Anotherembodiment is the treatment of psychiatric diseases and disorders byadministration of the compounds described herein. These compounds may beused to treat not only the disease or disorder but may also be used totreat the symptoms and effects of the disease or disorder. For example,a subject afflicted by schizophrenia may exhibit symptoms such asdelusions, ahedonia, and avolition.

EXAMPLES

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the invention to the particular features or embodiments described.

Example 1

This example describes the in vitro inhibition of heme aggregation byxanthone 2,3,4,5,6-pentahydroxyxanthone. Conditions: 25 mM Phosphatebuffer, pH 5.2, 1 hour incubation at 37° C. Further details of thisassay are described by Ignatushchenko et al. in Xanthones asantimalarial agents; studies of a possible mode of action. FEBS Lett.409, 67-73 (1997). With reference to FIG. 1, panel A, the left tubecontains heme alone; the center tube contains heme with the xanthone;and the right: tube includes heme with chloroquine.

With continued reference to FIG. 1, panel B illustrates thespectrophotometric titration curve of the heme:3,6-bis-ε-(N,N-diethylamino)-amyloxy xanthone (C5) complex. The apparentbinding constant was derived from the best fit (solid line) of a 1:1(heme dimer: C5) association model. Panel C includes images fromconfocal fluorescence microscopy of C5 and LysoTracker Red accumulationin a P. falciparum infected erythrocyte. Panels A and B show thefluorescence localization of C5 and LysoTracker Red, respectively. PanelC shows the brightfield transmission image of the infected cell overlaidwith the image from Panel A. Panel D shows the merged imagedemonstrating co-localization of C5 and LysoTracker Red.

Example 2

This example illustrates the ability of disclosed antimalarialacridones, such as 10-(ω-(N,N-diethylamino-alkyl)acridones to enhance CQpotency against MDR P. falciparum strains W2 and Dd2. The responsemodification index (RMI) was used to represent the IC50 of CQ in thepresence of a fixed concentration of the test acridones in comparison toCQ alone (RMI=IC50 of CQ with acridone/IC50 of CQ alone). The resultsare summarized in Table 1. An RMI of 1.0 indicates no change in IC50upon addition of a resistance reversal agent, >1.0 indicates antagonism,and <1.0 indicates chemosensitization and possible synergism.

TABLE I In vitro chemosensitization to chloroquine in the presence of 5μM acridone derivatives and selected known chemosensitizers. DrugChemosensitizer W2 Dd2 Calc'd combination Structure IC₅₀ ^(a)(nM)RMI^(b) IC₅₀ ^(a)(nM) RMI^(b) pK_(a) ^(c) Chloroquine (CQ) alone

290.0 — 100.2 — 10.3 6.3 CQ + A2

259.3 0.88 80.9 0.76 9.81 CQ + A3

60.9 0.21 33.4 0.31 10.28 CQ + A4

35.3 0.12 12.0 0.11 10.46 CQ + A5

30.5 0.10 11.3 0.11 10.53 CQ + A6

17.8 0.06 8.1 0.08 10.56 CQ + A8

39.5 0.14 15.5 0.16 10.65 CQ + A6-Cl

550.4 1.87 176.9 1.66 — CQ + verapamil

51.8 0.18 21.5 0.2 8.6 CQ + desipramine

29.0 0.10 10.0 0.10 10.4 CQ + chlorpheniramine

58.0 0.10 11.0 0.11 10.2 ^(a)IC₅₀ of CQ in combination with 5 μM ofselected drug. ^(b)MRI = IC₅₀ of CQ in combination/IC₅₀ of CQ alone.^(c)pK_(a) calculated with ChemSketch I-Lab

Acridones A2, A3, A4, A5, A6, A8, with a tertiary amine at the end ofthe carbon chain attached at the N-10 position, increased, in some casesdramatically, the sensitivity W2 and Dd2 to CQ. In the presence of 5 μMA6, the IC₅₀ of CQ was potentiated by over 90-fold in both of the MDRstrains to a level (17.8 nM, W2; 8.1 nM, Dd2) approximately equivalentto that observed for the CQ-sensitive strain D6 (15 nM). Acridonecongeners A3 to A6 were efficacious chemosensitizers, and disclosedacridone compounds A4 to A6 exhibited comparable or superiorchemosensitization, to the known agents verapamil, desipramine, andchlorpheniramine.

Example 3

This example describes the screening of disclosed antimalarial acridonesagainst the CQ-sensitive D6 strain and the MDR Dd2 strain of P.falciparum for in vitro antimalarial activity. The results of thescreening of exemplary acridones are recorded in Table 2.

Notably, the screened compounds were equally potent against D6 and Dd2.For most of the compounds, including T2 and T3.5, there was no evidenceof cytotoxicity at the highest tested concentration (25 μM), and lesscytotoxicity than CQ. All of the compounds illustrated in Table 3 areweak bases (pK_(a) 95-10.5), favoring accumulation of their charged forminside the acidic parasite digestive vacuole, particularly in the caseof the dibasic compounds. It is also noteworthy that the cLogP valuesare <5, indicating adequate aqueous solubility for realistic drugdelivery.

TABLE 2 In vitro assessment of antimalarial potency vs. sensitive &multidrug resistant strains of P. falciparum. IC₅₀ ^(b), Drug IC₅₀ ^(a),nM nM Name Structure D6 Dd2 MSLCS IVTI^(c) pK_(a) ^(e) 3-(2-diethylaminoethoxy)- 9-acridone (T1)

167 159 >25,000 >150  9.5 3-(2- diethylaminoethoxy)--6-chloro-9-acridone (T2)

18 17 >25,000 >1389  9.5 3-(2- diethylaminoethoxy)- 6-chloro-xanthone(TX2)

>2,500 >2,500 >25,000 NA  9.5 2-(2- diethylaminoethoxy)--6-chloro-9-acridone (T2.2)

106 123 >25,000 >235  9.5 3-(3- diethylaminoethoxy)- 9-acridone (T3)

60 60 >25,000 >417 10.1 3-(5- diethylaminoethoxy)- chloro-9-acridone(T5)

84 125 10,000 119 10.5 2-(2- diethylaminoethoxy)--6-chloro-10-(2-diethyl) aminoethyl)-9-acridone (T2.2.5)

79 86 >25,000 >316  9.8  9.5 3-(2- diethylaminoethoxy)--6-chloro-10-(2-diethyl)- aminoethyl)-9-acridone (T3.5)

5.0 58 >25,000 >500  9.7  9.5 Chloroquine

6.8 132 1,300 191 10.3  6.3 ^(a)The SYBR Green MSF assay was used forIC₅₀ determinations; the values are the average of 4 independentexperiments. ^(b)MSLCs = Murine splenic lymphocytes. Cytotoxicity wasdetermined by the Alamar Blue assay. ^(c)IVTI = ratio of cytotoxicityIC₅₀ (vs. MSLCs) to antimalarial IC₅₀ against D6. ^(e)pK_(a) values werecalculated with ChemSketch I-Lab

As shown in Table 3 and Table 4, selected soluble acridone derivativeswere screened for antimalarial activity against the CQ-sensitive D6 andthe MDR Dd2 strains of P. falciparum.

TABLE 3 In vitro intrinsic antimalarial activity, cytotoxicity,heme-binding affinity, and physical properties of selected acridoneswithout N-10 substitution. IC₅₀* (nM) P. falciparum IC₅₀ (nM)^(a)Heme-binding Drug Structure D6* Dd2* MSLCs cLogP^(b) K_(a) ^(c) (10⁴M⁻¹) T1

167 159 >25,000 3.2 TBD T2

26 33 >25,000 3.8 4.3 T2.1

240 420 >25,000 3.8 TBD T2.2

70 126 >25,000 3.8 5.4 T3

104 109 >25,000 3.9 TBD T4

398 759 >25,000 3.8 TBD T5

76 61 >25,000 4.7 TBD T6

270 390 TBD 3.4 TBD T7

90 49 TBD 3.2 TBD T8

12 19 TBD 3.9 TBD T9

56 63 TBD 2.7 TBD TX2

>2,500 >2,500 >25,000 3.9 TBD CQ^(†)

8.4 124 1,300 3.1 5.5 ATV^(†)

0.1 0.1 >25,000 3.7 — TBD: to be determined *All IC₅₀ were assessed byMSF assay; D6: CQ sensitive; Dd2; MDR, Old World genetic background; Seetext for details. ^(a)MSLCs = murine splenic lymphocytes. Cytotoxicitywas determined by the Alamar Blue assay. ^(b)LogP values were calculatedwith ChemDraw Ultra 8.0 software.

TABLE 4 In vitro antimalarial activity, cytotoxicity, and heme-bindingaffinity, of selected acridones. IC₅₀ (nM) P. falciparum IC₅₀ (nM)^(a)Heme-binding DRUG Structure D6^(*) Dd2^(*) MSLCs K_(a) ^(b) (10⁴ M⁻¹)T3.5

44 77 >25,000 7.1 T2.1.5

33 84 >25,000 TBD T2.2.5

56 133 >25,000 TBD T4.5

47 100 >25,000 TBD T5.5

76 109 8,900 TBD T7.5

17 20 TBD 4.3 T8.5

50 116 TBD TBD T9.5

33 60 TBD TBD T10.5

637 595 TBD TBD T11.5

196 359 TBD TBD QN

19 87 TBD TBD TBD: to be determined. *^(,a,b)See Table 2 footnote fordetails. ^(†)QN: quinine.

Example 4

This example describes evaluation of the in vivo antimalarial efficacyof the disclosed acridones against CQ-sensitive P. yoelii (strain K) infemale CF-1 mice. Illustrated in FIG. 2, are the results of a 4-daysuppressive test with acridone T2(3-(2-diethylaminoethoxy)-6-chloro-9-acridone)) in PBS administeredintragastrically (ig) by gavage one hour after infection and then oncedaily, 24, 48 and 72 hours later. The T2 acridone exhibited excellentparasite inhibition and oral bioavailability with an ED₅₀ of 27.1 mg/kg,and ED₉₀ of 42 mg/kg. A dose of 200 mg/kg/day T2 completely suppressedparasitemia as assessed 24 hours after the last dose, and all of thefour mice in that group remained parasite-free to the end of theobservation period.

Example 5

This example describes the assessment of curative efficacy of acridoneT2 (2-(2-diethylaminoethoxy)-6-chloro-9-acridone) in an in vivo modelwherein treatment is started only after infection is patent (i.e.,parasitemia is evident). For these studies, 5×10⁶ P. yoelii (K)parasites were injected intravenously (iv) into naïve mice. Forty-eighthours later, parasitemia rose to 3-5%, and T2 was givenintraperitoneally (ip) at 16 mg/kg, 64 mg/kg, and 256 mg/kg or ig (256mg/kg only) once daily for three consecutive days. Blood smears werecollected 24 hours after the last treatment. T2 brought about a rapidreduction in parasitemia in this regimen, with ED₅₀ (ip) of 38 mg/kg/dayand ED₉₀ (ip) of 66 mg/kg/day. Enteral and parental dosing could only becompared at 256 mg/kg/day, but at that dose there was equal efficacy.There was a >99.9% reduction in parasitemia in all animals given 256mg/kg/day (both ig and ip), and all were without evident parasitemia at30 days. T2 exhibited similar efficacy by gavage against patent P.berghei (ANKA) infection (ED₅₀=55 mg/kg/day).

The single-dose efficacy of acridone compounds also was assessed in vivousing ig treatment after patent infection. Test compounds wereadministered as a single dose at 100 mg/kg (30 mg/kg for CQ) 24 hoursafter P. yoelii infection, and blood smears were collected 48 and 72hours post-treatment. The results for 3 acridone derivatives as well asCQ are displayed in FIG. 3. Compared with controls, T2 (HCl salt), T3.5(di-HCl salt) and CQ each reduced parasitemia by more than 80% at 48hours, and greater than 90% reduction persisted at 72 hours after T2administration. Due to the difference in the molecular weight betweenthe salts of T2 and T3.5, the apparent superiority of T2 over T3.5 at 72hours may only reflect a lack of molar equivalence in dosing, ratherthan pharmacokinetic and/or pharmacodynamic advantages of T2). Theresults also validate this single-dose rodent model as a useful tool forprimary screening of in vivo antimalarial activity, and for assistingthe process of lead optimization of acridone derivatives. Patentinfection before treatment, single-dose treatment and serialobservations provide valuable information with minimal resource use thatshould be predictive of ultimate efficacy, curative effect,bioavailability, toxicity, and pharmacokinetic/pharmacodynamicproperties.

It is noteworthy that the highest doses in the in vivo tests (200mg/kg/day×4 days and 256 mg/kg/day×3 days) were well tolerated.Considering the ED₅₀ values, the lack of evident toxicity forecasts afavorable in vivo therapeutic safety index for acridone derivatives.

Example 6

This example describes the evaluation of chemosensitization andsynergistic effects produced by administration of the disclosedacridones in combination with other chemotherapeutic agents, includinganticancer, antibiotic, antiviral, and antiparasitic agents, such asantimalarial agents.

Antimalarial drug combinations can be assessed in a number of ways. Forexample, the response modification index (RMI) can be calculated by thefollowing formula: RMI=IC₅₀ of drug A in the presence of drug B/IC₅₀ ofdrug A alone, where drug A is an existing antimalarial and B is a singlesub-inhibitory concentration of the acridone candidate. An RMI less than1.0 represents chemosensitization and possible synergy. A second methodof evaluating combination therapies is Fractional InhibitoryConcentration, (e.g., FIC₅₀=[IC₅₀ of Drug A in the presence of a fixedamount of Drug B]/[IC₅₀ of Drug A alone]). RMI and FIC are helpfulmeasures for side-by-side comparison of multiple chemosensitizer drugsat fixed concentrations, but neither distinguishes between additive andsynergistic effects, or describes effects over a wide concentrationrange. Standard FIC indices (FIC of Drug A+FIC of Drug B) distinguishsynergistic, additive and antagonistic interactions, but are dependenton the correct estimation of the expected effect of the fixed test drug.

In one embodiment, synergism is assessed herein using fixed-ratiocombinations that provide information over a wide range of drugconcentrations. In this method, the two drugs are mixed in anappropriate ratio, serial dilutions of the combined solution are usedfor testing, and results compared to each drug alone. Specifically, todetermine synergy, the acridone is tested in combination with a seconddrug using a modification of the fixed-ratio method described byFivelman et al. (See, Fivelman et al., Antimicrob. Agents Chemother. 48,4097-4102 (2004) and Winter et al., Antimicrob. Agents Chemother. 41,1449-1454 (1997), both references incorporated herein, in theirentirety, by reference). In this method, after IC₅₀ determination forall test drugs, stock solutions were prepared of each drug atconcentrations such that the final concentration in the 96-well platedrug susceptibility assay after 4-5 2-fold dilutions will approximatethe IC₅₀. If these stock solutions were termed Drug A and Drug B, thensix final stock solutions were prepared from this initial stock: Drug Aalone. Drug B alone, and volume:volume mixtures of Drug A and B in thefollowing ratios: 4:1, 3:2, 2:3, and 1:4. Two-fold dilutions of each ofthe six final stock solutions were done robotically across a 96-wellplate in quadruplicate. Subsequent steps were typical of standarddrug-susceptibility methods. Initial data analysis yielded the intrinsicdose-response curve for each drug alone, and four different fixed:ratiocombination dose-response curves. The data pointed from all six curveswere then analyzed using Calcusyn software (commercially available fromBioSoft, Cambridge. UK) for determination of synergy. Output from thisanalysis included FIC values used to plot isobolograms as well as arigorously determined synergy index, termed the combination index (CI)(Calcusyn). FIC₅₀ isobolograms, CI's and tabulation of IC₉₀ componentdrug concentrations were reported.

Using this method, T3.5(3-(2-diethylaminoethoxy)-6-chloro-10-(2-diethylaminoethyl)-9-acridone)was evaluated in combination with the known antimalarial quinolineschloroquine, quinine, amodiaquine and mefloquine. The results aredepicted in four isobolograms in FIG. 4.

The combinations of the acridones T3.5 and T2 with quinine were assessedfor synergism. The results are charted in the isobologram of FIG. 5.With reference to FIG. 5, values below the diagonal (additive) lineindicate synergism, demonstrating synergism between quinine and T3.5,but not quinine and T2.

FIG. 6 is an isobologram charting the efficacy of quinine in combinationwith T3.5 in vitro against CQ-sensitive (D6) and MDR (Dd2) P.falciparum. Values below the diagonal (additive) line indicate synergismbetween quinine and T3.5 against both strains. The results illustratedin FIG. 6, demonstrate 90% growth inhibition of quinine-resistant Dd2was achieved at low concentrations (20-200 nM T3.5 in combination with7-70 nM quinine), a significant advance toward clinical value.

Example 7

This example describes visual evidence that acridones disclosed hereinform soluble complexes with heme under mildly acidic conditions. Asdescribed in Example 1, tricyclic xanthones formed soluble complexeswith heme, preventing heme precipitation in vitro. Employing the sameassay conditions, heme was mixed with T2 or T3.5 under mildly acidicconditions (25 mM phosphate buffer, pH 4.8) and monitored. As in thecase of the xanthones, after the first 30 minutes at room temperature itwas evident that T2 and T3.5 form soluble complexes with heme under thepH conditions that exist in the digestive vacuole. Both T2 and T3.5prevent the formation of heme aggregates, a process that is normallyspontaneous at this pH. Without being limited to theory, it is believedthat for some compounds inhibition of heme aggregation in vitro can becorrelated with inhibition of hemozoin formation in vivo. Thisstraightforward in vitro test can be used to provide a preliminaryassessment of antimalarial potential of antimalarial acridones. It isnoteworthy that CQ fails to prevent heme aggregation in this assay, aresult probably reflecting substantive differences in affinity, bindinggeometry and other factors.

Example 8

This example describes a general synthetic approach to the disclosedacridone compounds. The acridone core is assembled in according toScheme 1 using the classic Ullman copper-mediated coupling reaction.With reference to the variable groups, X, Y, R¹ and R are as disclosedherein above, and Z represents H or a protected functional group, suchas a masked group G. Ullman coupling of a suitable 2-chlorobenzoic acidderivative 10 with an appropriately substituted aniline derivative 20gives diphenyl amine 30. Ring closure under acidic conditions yieldstricyclic acridone 40. Various acridone derivatives can be prepared fromtricyclic acridone 40, for example, the 10-position nitrogen isalkylated with alkyl halides to produce N-substituted acridone 50.

Compounds 40 and 50 can be further functionalized. For example when oneof YR¹Z represents an alkoxy group, such as a methoxy group, the methoxygroup can be cleaved to yield free phenols 60 and 70, for example as setforth in the Scheme 2. With continued reference to Scheme 2, suchphenolic compounds can be further derivatized, for example compounds 60and 70 can be alkylated with a haloalkyl group, such as a dibromoalkylgroup compound to give compounds 80 and 90, respectively. Acridonederivatives 80 and 90 can be further functionalized, for example byalkylating a secondary amine to give compounds 100 and 110.

T 3.5(3-(diethylamino-ethoxy)-6-chloro-N-10-diethylaminoethyl-9-acridone) wasprepared according to the schemes above, with the final product obtainedby reacting 3-hydroxy-6-chloro-9-acridone with 2 equivalents of(2-chloro-ethyl)-diethyl-amine (as the hydrochloride salt) in acetonewith potassium carbonate at reflux for 12 hours. After this period thesolvent was removed in vacuo and the residue was taken up in hexanewhereupon it crystallized on standing and evaporation, and in highyield, ca. 50 to 100% of theoretical yield.

Similarly, T2 (3-diethylamino-ethoxy)-6-chloro-9-acridone) was preparedaccording to the schemes above, with the final product being obtained byreacting 3-hydroxy-6-chloro-9-acridone with one equivalent of(2-chloro-ethyl)-diethyl-amine (as the hydrochloride salt) in ethanoland in the presence of a 1.5 equivalent excess of potassium hydroxideand heated at reflux for 12 hours. After this period the reaction vesselwas taken to dryness and the residue was taken into a mixture of ethanoland water. Upon cooling the desired product fell out of solution and wasfiltered and dried in this high yielding reaction, ca. 50 to 100% ofexpected yields.

Example 9

This example describes evaluation of acridones in rodent models ofblood-stage malaria. Initial testing of the in vivo antimalarialefficacy of gavage-administered T2 (HCl salt) against CQ sensitive P.yoelii (K) in the standard 4-day suppressive test (1) revealed excellentparasite inhibition (ED₅₀ 27.1 mg/kg, ED₉₀ 42 mg/kg, completesuppression at 200 mg/kg/day).

In an adaptation of the standard 6-day Thompson test, three once-dailydoses were administered to mice starting 2 days after infection, whenparasitemia was ˜3%, and efficacy was measured by determiningparasitemia from blood smears obtained 1 day after the third and finaldose. Using gavage (ig) dosing in this model, the ED₅₀ and ED₉₀ of T2,T3.5, and QN are comparable against P. yoelii (K) (FIG. 8). T2 has alsobeen assessed after intraperitoneal (ip) dosing, with comparable results(ED₅₀ and ED₉₀=38 and 66 mg/kg/day, respectively), and against P.berghei (ANKA) (55 and 80 mg/kg/day, respectively). T2 testing at 256mg/kg/day resulted in long-term cure.

Example 10

This example evaluates the prophylactic efficacy of the acridone T2. T2was evaluated in a murine P. yoelii sporozoite-induced malaria model todetermine prophylactic efficacy.

1 Mice were treated with 160 mg/kg ig, on days −1, 0, and 1 withinjection of 250,000 sporozoites on day 0. Blood smears on days 6, 11,and 14 showed all 5 treated animals to be parasite-free, a resultcomparable to that of primaquine in the same model. No toxicity wasobserved.

Drug doses administered include 300 mg/kg in a single dose (T2, T2.2,T3.5, T7.5, T8.5, T9.5), 256 mg/kg/day×3 days (T1, T2), and 200mg/kg/day×4 days (T2, T3.5). Other than 10% weight loss in animalsreceiving T2 at 256 mg/kg/day×3 days, there has been no general toxicitynoted in mice treated with any of the candidate acridones, at any dose,and our initial cured animals are alive and well nearly a year aftertreatment. An in vitro screen for cytotoxicity in murine spleniclymphocytes shows that the IC₅₀ of CQ is 1.3 μM, whereas the IC₅₀ ofmost candidate acridones described in this proposal exceed 25 μM.

Example 11

This example provides an in vitro assessment of T2 and T3.5 activity ina model using cloned biogenic amine transporters. Unlike tricyclicantidepressants and cocaine positive controls, the acridones had nosignificant affinity for serotonin, dopamine, or norepinephrinetransporters (Table 5).

TABLE 5 Effects of drugs on inhibition of radioligand [¹²⁵I]RTI- 55binding to the recombinant human dopamine (hDAT), serotonin (hSERT), andnorepinephrine (hNET) transporters stably expressed in human embryonickidney (HEK) cells. [¹²⁵I]RTI-55 [¹²⁵I]RTI-55 [¹²⁵I]RTI-55 BindingBinding Binding HEK-hDAT HEK-hSERT HEK-hNET Compound K_(i) (nM) K_(i)(nM) K_(i) (nM) T2 >10,000 >10,000 5848.50 T3.5 >10,000 2292.72 >10,000Mefloquine >10,000 90.02 3875.09 Cocaine* 296.12 198.72 655.94 *Cocainewas used as a positive control.

Example 12

This example provides morphological changes in acridone treatedparasites. In vitro human PRBCs (P. falciparum D6, Dd2), ex vivo mousePRBCs (P. yoelii (Kenya) and in vivo mouse PRBCs (P. yoelii (Kenya), P.berghei [ANKA gfp+,luc+]) all demonstrate failure of acridone-exposedparasites to progress from trophozoite to schizont stage. Compared todrug-free controls, T1-treated late trophozoites appear pale stainingwith loss of intracellular definition and fragmented hemozoin (FIG. 9).When drug treatment followed high parasitemia (as in FIG. 9), many suchabnormal parasites were evident 24 hr after cessation of drug treatmentbut then were rapidly cleared over the following 24-48 hours, indicatingnon-viability. These preliminary evaluations of blood smears by lightmicroscopy indicate that acridones cause alterations in parasitemorphology and viability.

P. falciparum Dd2 MDR parasited red cells were synchronized to the ringstage by sorbitol lysis and incubated the cells in the presence orabsence of 300 nM T3.5. As compared to controls after exposure to T3.5,the PRBC cytoplasm was more basophilic and visible hemozoin was markedlydiminished. FIG. 10 shows images of control vs. drug treated parasitesafter 36 hours of incubation. P. yoelii infected mice treated with T3.5harbor PRBCs that lack hemozoin. Taken together these observations areconsistent with the notion that T3.5 blocks hemozoin formation ininfected cells.

Example 13

This example shows a pattern of synergy with quinoline antimalarials: aproperty of the T3.5 type of acridones. In vitro isobolar analysisdemonstrates that T3.5 is a potent chemosensitizer, synergistic incombination with CQ. QN or amodiaquine, but not with mefloquine, againstthe MDR Dd2 clone while there is no evidence of synergy in the additiveinteraction between T2 and QN (see FIG. 1). Screening of T3.5 and otheracridones by determination of RMI indicates chemosensitizing actionthroughout the group (see Table 6).

TABLE 6 In vitro chemosensitizing effects of acridones against CQS (D6)and MDR (Dd2) strains of P. falciparum. D6 Dd2 Drug IC₅₀ IC₅₀Combination (nM) RMI^(a) (nM) RMI^(a) QN alone 20 — 87 — QN + 7.5 0.3811 0.13 50 nM T3.5 QN + 7.1 0.36 8.0 0.09 50 nM T2.1.5 QN + 5.4 0.28 120.13 50 nM T2.2.5 QN + 4.2 0.22 9.6 0.11 50 nM T4.5 QN + 4.0 0.20 7.80.09 50 nM T6.5 QN + 21 1.1 85 0.98 50 nM VP^(b) CQ alone 8.4 — 124 —CQ + 3.7 0.44 32 0.26 50 nM T3.5 CQ + 6.7 0.79 47 0.38 50 nM T2.1.5 CQ +3.1 0.37 32 0.26 50 nM T2.2.5 CQ + 2.9 0.35 39 0.31 50 nM T4.5 CQ + 2.00.24 28 0.23 50 nM T6.5 CQ + 8.3 0.99 118 0.95 50 nM VP^(b) QN: quinine

RMI: IC₅₀ of QN (or CQ) in combination with chemosensitizers/IC₅₀ of QN(or CQ) alone. ^(b)VP: verapamil.

indicates data missing or illegible when filed

Example 14

This example provides a demonstration of in vivo synergism between T3.5and quinine. In vivo synergism was assessed using the 6-day Thompsontest in mice infected with QN-sensitive P. yoelii (K), comparing theefficacy of T3.5 alone, QN alone and T3.5:QN combinations (Table 7).Dosages were established using a rigorous fixed-ratio combination dosingprotocol and results were assessed using Calcusyn software to determinesynergy. As in the case of the in vitro combination, there was evidentin vivo synergism. At the ED₅₀, ED₇₅ and ED₉₀ effect levels, theCombination Index (CI) was <0.6, indicating definite and significantsynergy. More clinically relevant than this measure of synergy, the dosecombination resulting in 90% inhibition of growth suggests thatsubstantial dose reductions of the individual drugs can be achieved.Alone, the ED₉₀ of T3.5 and QN were 88 and 85 mg/kg/day, respectively;only 24 mg/kg/day of each in combination produced the same result.

TABLE 7 Synergism between T3.5 & quinine in vivo vs. patent infectionsof P. yoelii (quinine sensitive) in mice. Dose (mg/kg/day) T3.5 QuinineT3.5:Quinine Effect alone alone Combination ED₅₀ 56 39 14:14 ED₇₅ 70 5719:19 ED₉₀ 88 85 24:24

Example 15

This example investigates the metabolic stability and fate of T3.5 inthe presence of NADPH and a murine hepatic S9 fraction. Hepatic S-9fractions were incubated with 50 μM T3.5 in the presence of NADPH (1 mM)for 10, 30 and 60 min. HPLC was conducted with a Hypersil Gold columnthat is stable at high pH. As a result, a solvent system at pH 10.5 wasused and excellent peak shape was obtained for T3.5 (calculated pKa of9.5). Despite the high pH, T3.5 (a retention time of 18.5 min in PanelA. FIG. 12) exhibited an abundant molecular ion in the positive mode[M+H]⁺ at m/z 444.2. The full scan mass spectrum had the isotopic ratioexpected for the chlorine substituted acridone ring. FIG. 12 shows theextracted ion chromatograms for a series of possible P450-dependentmetabolites of T3.5 produced in the S-9 incubation with NADPH. The mostabundant metabolites were de-ethylated products monitored at m/z 416.2(loss of m/z=28) with retention times of 17.3 and 17.7 min. The twounresolved peaks most likely represent de-ethylation of the tertiaryamine on each side chain.

A second de-ethylation also occurred with the prolonged incubation asevidenced by the peak at 17.5 min when monitored at n/z 388.1 (loss ofm/z=56). Another dealkylated metabolite was monitored at m/z 345.1 andcorresponds to the loss of the triethylamine side chain (loss of m/z=99)from the acridone ring. The dealkylation could occur from either thearyl ether side chain or from the acridone ring nitrogen. The laterwould result in the production of the active compound T2. Extensivede-ethylation suggests that if the secondary amine metabolite is active,the pharmacodynamic half-life could be much longer than the parentcompound as active metabolites would be produced. In addition to thedealkylated products, a hydroxylated metabolite was also detected bymonitoring at m/z 460.2 (addition of m/z=16). The hydroxylation couldoccur on the acridone ring or on one of the primary carbons on theethylamine side chains. Further MS/MS experiments will help establishthe site of hydroxylation.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

1-56. (canceled)
 57. A compound according to the formula

or a pharmaceutically acceptable salt thereof; wherein R is H, —R²NR³R⁴or —R¹G; X is H, halogen, haloalkyl, OR⁵ or —YR¹G; n is 0-4; Y is H,—CH₂, —CH₂O—, O, —N(R⁶)— or S; R⁵ is lower alkyl, haloalkyl or —R⁷NR⁸R⁹;R¹, R² and R⁷ independently are optionally substituted alkyl; G is—NR¹⁰R¹¹, halogen or fluoroalkyl; R³, R⁴, R⁶, R⁸ and R⁹ independentlyare H, lower alkyl, or aralkyl; R¹⁰ and R¹¹ independently are H, loweralkyl, aralkyl or together form an aliphatic or aromatic ring optionallyincluding one or more additional heteroatoms; and when n is 0, R is—R¹G.
 58. A method for inhibiting or treating a parasitic infection in asubject, comprising administering to a subject a therapeuticallyeffective amount of the compound of claim
 57. 59. A method forinhibiting the growth of Plasmodium sp., comprising contacting at leastone Plasmodium sp. parasite with a growth inhibitory amount of thecompound of claim 57.